Four New Species of Hemileccinum (Xerocomoideae, Boletaceae) from Southwestern China

The genus Hemileccinum belongs to the subfamily Xerocomoideae of the family Boletaceae. In this study, phylogenetic inferences of Hemileccinum based on sequences of a single-locus (ITS) and a multi-locus (nrLSU, tef1-α, rpb1, rpb2) were conducted. Four new species, namely H. abidum, H. brevisporum, H. ferrugineipes and H. parvum were delimited and proposed based on morphological and molecular evidence. Descriptions and line-drawings of them were presented, as well as their comparisons to allied taxa. Our study shed new light on the recognition of the genus. The pileipellis of the species in this genus should mostly be regarded as (sub)epithelium to hyphoepithelium, because the pileipellis of most studied species here is composed of short inflated cells in the inner layer (subpellis) and filamentous hyphae in outer layer (suprapellis). The basidiospores of the studied species, including the type species, H. impolitum, have a warty surface.


Introduction
The genus Hemileccinum Šutara was created based on the species H. impolitum (Fr.) Šutara as the type, and H. depilatum (Redeuilh) Šutara [1]. These two species were both originally placed in the genus Boletus L. [2] and later were transferred to the genus Leccinum because of the lateral stipe stratum of the leccinoid type which is predominantly anticlinally arranged, breaking up into characteristic fascicles of hyphae ending in elements of the caulohymenium during growth of the stipe [1,3,4]. However, molecular phylogenetic analyses indicated that these two species are very distant from the species of both Leccinum and Boletus, but similar to the species of Xerocomus; thus they were accordingly transferred to Xerocomus [5,6]. Based on the previous molecular evidence and his own further morphological observations, Šutara established the genus Hemileccinum to accommodate these two species. He emphasized that Hemileccinum was diagnosed by the anatomical structure of the peripheral stipe layers having a lateral stipe stratum of the leccinoid type, which distinguished this genus not only from all the other species belonging to the Xerocomus s.l. but also from those in the genus Boletus [1]. Wu et al. confirmed the monophyly of Hemileccinum and found an additional diagnosed character of this genus, namely the irregularly warty basidiospores under SEM [7,8]. Meanwhile, the genus Corneroboletus N.K. Zeng & Zhu L. Yang was confirmed as a synonym of Hemileccinum due to the similar basidiospore ornamentation and the closely phylogenetic relationship [8].
During past fungal investigations in southwestern China, we encountered four potential new species of Hemileccinum. Our aim in this study is to clarify their molecular phylogenetic positions and to delimit them based on morphological data and molecular evidence.

Sample Collection and Morphological Study
In total, seventeen collections were examined in this study, which were collected from the Yunnan Province of southwestern China during the years 2007-2017 ( Figure 1). The macroscopic characters of the specimens were described based on fresh basidiomata, and the dried specimens were deposited in the Cryptogamic Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS). Color codes of the form "5C4" indicate the plate, row, and color block from Kornerup and Wanscher [17]. For microscopic observation, a ZEISS Axiostar Plus microscope (Oberkochen, Germany) was used and the dried specimens were revived in 5% KOH solution or distilled water. Moreover, Melzer's reagent was applied to test color reactions of the tissue fragments to the solution. Microscopic studies follow Li et al. and Zhou et al. [18,19]. In the descriptions of basidiospores, the abbreviation [n/m/p] means n basidiospores measured from m basidiomata of p collections. The range notation (a)b-c(d) stands for the dimensions of basidiospores in which b-c contains a minimum of 90% of the measured values while a and d in the brackets stand for the extreme values. Q is used to imply "length/width ratio" of a basidiospore in side view; Q m means average Q of all basidiospores ± sample standard deviation. To observe basidiospore ornamentations, a ZEISS Sigma 300 scanning electron microscope (SEM) (Oberkochen, Germany) was used. Genera are abbreviated as follows: H. for Hemileccinum, Ca. for Castanopsis, C. for Castanea, L. for Lithocarpus, P. for Pinus, Q. for Quercus and Rug. for Rugiboletus.

Molecular Procedures and Phylogenetic Analyses
Total genomic DNA was obtained with the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) according to the manual from material dried with silica gel. A total of five nuclear loci were sequenced, including the internal transcribed spacer (ITS), the large subunit of nuclear ribosomal RNA gene (nrLSU), the polymerase II subunit one (rpb1) gene, the second largest subunit of RNA polymerase II (rpb2), and the translation elongation factor 1-α gene (tef1-α). The primer pairs of ITS1/ITS4 [20,21], LROR/LR5 [22,23] were used for amplifying ITS, nrLSU, respectively. The primer pairs used for amplifying the rpb1, rpb2, tef1-α, followed those in Wu et al. [7]. PCR was performed in a total volume of 25 µL containing 1 µL forward primer, 1 µL reverse primer, 9.5 µL nuclease-free H 2 O, 12.5 µL BlasTaq TM 2×PCR MasterMix (abm, Richmond, VA, Canada) and 1 µL DNA template. PCR protocol was as follows: pre-denaturation at 95 • C for 5 min, followed by 35 cycles of denaturation at 95 • C for 60 s, 52 • C for 60 s, and 72 • C for 80 s, and then a final elongation at 72 • C for 8 min was included. The PCR products were purified with a Gel Extraction and PCR Purification Combo Kit (Spincolumn) (Bioteke, Beijing, China), and then sequenced by ABI-3730-XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA) by using the same primer pairs as in the PCR amplification for sequencing.

Phylogenetic Analyses
We used BLAST to compare the obtained sequences of the newly collected materials with those in the GenBank database. The BLAST results were used to predict the phylogenetic relationship between the newly collected specimens and known species and indicated that the new materials were genetically similar to the other species of Hemileccinum. In this study, two datasets were produced, the ITS dataset, and the combined nrLSU, tef1-α, rpb1 and rpb2 dataset. The ITS sequences of Hemileccinum species from China were used to infer relationships of Chinese species with those from Europe, North America and East Asia. In the analysis of ITS dataset, Phylloporus rubrosquamosus N.K. Zeng [24][25][26]. The combined dataset was mainly used to infer phylogenetic relationships and systematic positions of the Chinese species. In the multigene phylogenetic analysis, including all known genera in the subfamily Xerocomoideae were included. We screened the relevant sequences deposited in GenBank, which were mainly submitted by Wu et al. [7,8], Gelardi et al. [27], Zhu et al. [26], Zeng et al. [24], Neves et al. [28]. We collected a total of 13 ingroup species of 8 known genera within Xerocomoideae and 2 outgroup species outside Xerocomoideae but in the Boletaceae. Detailed information of the voucher specimens is given in Table 1.
The sequences were assembled with SeqMan implemented in Lasergene v7.1 (DNAS-TAR Inc., Madison, WI, USA), and then aligned by using MAFFT v7.310 [29]. The software Bioedit v7.2.5 [30] was used to check aligned matrices. To assess any potential conflicts in the gene tree topologies for these five loci, single-locus matrices were analyzed using maximum likelihood (ML) in RAxML v8.0.20 [31]. Sequences of the loci without conflicts were then concatenated using Phyutility 2.2 [32,33] for further phylogenetic analyses. The best-fitted substitution model for each gene was determined through MrModeltest v2.4 [34] by using Akaike Information Criterion (AIC). GTR + I + G was inferred as the best-fit model for the nrLSU, tef1-α, rpb1 and ITS selected according to the AIC in MrModeltest v2.4 [34]. SYM + I + G was selected as the best model for rpb2. For the ultimate phylogenetic analyses, Maximum Likelihood (ML) analysis and Bayesian Inference were conducted by RAxML v8.0.20 [31] and MRBAYES v3.2.7 [35], respectively. The parameters of RAxML were set as defaults with 500 bootstrap replicates, except the substitution model which was set as GTRGAMMAI.
BI analyses were conducted with two independent runs of one cold and three heated chains. Runs were performed for 2 million generations, and trees sampled every 100 generations. The convergence was determined with the average standard deviation of split frequencies (<0.01) Chain convergence was determined using Tracer v1.5 to confirm sufficiently large ESS values (>200). The sampled trees were subsequently summarized by using the "sump" and "sumt" commands with a 25% burn-in [31,35]. The Bayesian posterior probabilities (BPP) of internodes were estimated based on the majority rule consensus with the remaining trees.

Molecular Phylogenetic Analysis
A total of 79 sequences, including 16 for ITS, 12 for nrLSU, 17 for tef1-α, 17 for rpb1, and 17 for rpb2 were newly generated in the present study and aligned with sequences downloaded from GenBank and previous studies. Sequences retrieved from GenBank and obtained in this study were listed in Table 1. ML and BI analyses of the ITS dataset resulted in almost identical topologies and thus only the tree inferred from ML analysis was displayed (Figure 2). Our phylogenetic analyses indicated that Hemileccinum formed a monophyletic group with evident support (MLB/BPP = 100%/1.0). Eight phylogenetic species of the genus Hemileccinum were retrieved, and four of them could be new to science. According to the four single-locus phylogenetic analyses, no strongly supported (>70% of ML) conflict of topologies was observed. Therefore, sequences of the four DNA loci were concatenated for the final analysis. ML and BI analyses of the concatenated data set resulted in almost identical topologies and thus only the tree inferred from ML analysis was displayed ( Figure 3). Our molecular phylogenetic analysis indicated that Hemileccinum is a monophyletic genus with high statistic supports (BP = 98%, PP = 1). Thirteen phylogenetic species of the genus Hemileccinum were retrieved, and four of them could be new to science. By further morphological examinations of the related specimens of those four potential new species, we verified their taxonomic statuses of new species. For detailed information of each species, see below.  Diagnosis: Hemileccinum albidum is distinguished by the combination characters of the even pileus, and the whitish, nearly smooth stipe, with only small, granular scales at the base.
Description: Basidioma stipitate-pileate, small to medium-sized. Pileus 3-10 cm diam, clavate to planate, surface rugose, slightly subtomentose, dry, yellowish brown (5E5), olive brown (4E5-6) to dull brown (5E8-5F8), context cream to yellowish (2A4-5), unchanging when bruised. Hymenophoral surface and tubes concolorous, yellow (1A2-1A3) to ochreous (5B7-5C7), unchanging when bruised, pores roundish, 1.5-2.5/mm; tubes 4-6 mm long, unchanging when injured. Stipe 4-10 cm long, 1-2 cm wide, subcylindrical, surface yellowish to yellow at upper part, lower part pale red-brown of stipe pileus; covered with longitudinal striations and densely dotted scales, context cream (1A2) to yellowish, unchanging when bruised. Basal mycelium cream.  Notes: Hemileccinum ferrugineipes is characterized by its rugose pileus and small, dense, dotted scales on the reddish-brown stipe. Phylogenetically, the American species H. subglabripes is close to H. ferrugineipes, but differs from it by its fairly long and slender, nearly smooth stipe [9,10,[43][44][45]. Morphologically, H. ferrugineipes is similar to Rugiboletus extremiorientalis (Lj.N.Vassiljeva) G. Wu & Zhu L. Yang and H. hortonii in the rugose pileus and dense scales on the stipe [44][45][46][47]. However, H. ferrugineipes differs from Rug. extremiorientalis, originally described from subtropical Yunnan, China, by its reddish slightly densely scaled surface of the stipe and hyphoepithelium pileipellis. H. ferrugineipes differs from H. hortonii, originally described from Illinois, USA, by its tightly wrinkled pileus and the stockier stipe [9,44,45]. Ecologically, H. ferrugineipes occurs under trees of Fagaceae in subtropical regions; H. hortonii is scattered or in groups on the ground under mixed deciduous woods, occasionally under conifers; H. hortonii is rather rare and might be found in eastern North America, west to Michigan [44,45]  on acidic, wet, fertile soils; rather rare; fruiting in July in southeastern Yunnan between 1200 and 1300 m altitude. Notes: Hemileccinum parvum is morphologically similar to H. subglabripes because of the slightly wrinkled pileus and the slender stipe [9,48], However, H. subglabripes, originally described from the USA, differs from H. parvum by the nearly smooth stipe of the latter covered with branny particles on the stem which are pale and easily overlooked, and the larger basidioma. Our data show that H. parvum is phylogenetically close to H. rubropunctum, but the latter differs by its longer stipe and the red scales on it [10,44,45]. Ecologically, H. parvum occurs under trees of Fagaceae in subtropical southeastern Yunnan; H. subglabripes inhabits mixed deciduous trees, sometimes under spruce in eastern and particularly northern North America; and H. rubropunctum grows in mixed woods with oak or chestnut in northeastern North America [44,45] (Appendix A).

Discussion
The genus Hemileccinum Šutara is geographically widely distributed, but its species diversity is poorly known. In Asia, only two species have been previously reported with molecular evidence. One is H. indecorum from tropical areas, and the other is H. rugosum from subtropical Yunnan, China [8,15]. In this study, four new species in China were recognized and delimited. They are well-supported by molecular phylogenetic and morphological evidence. The host specificity, altitude and edaphic factors seem to be important for determining the distribution of different species of Hemileccinum. Our newly described species are distributed in the broad-leaved and mixed forests in southwestern China. Hemileccinum albidum and H. brevisporum are distributed on high altitudes: between 1700 and 2500 m a.s.l., while H. ferrugineipes: 1200-1700 m a.s.l., and H. parvum: 1200-1300 m a.s.l. Hemileccinum albidum, H. ferrugineipes and H. parvum are found in subtropical forests and associated with plants of the family Fagaceae (Castanopsis ceratacantha, Ca. rufescens, Ca. ferox, Ca. hystrix, Ca. calathiformis; Castanea henryi, C. mollissima; Cyclobalanopsis xanthotricha; Lithocarpus xylocarpus, L. hancei, L. mairei, L. glabra, L. bonnetii; Quercus griffithii, Q. fabri, Q. variabilis, Q. marlipoensis), growing mostly on acidic soils. However, H. ferrugineipes can also be found in slightly alkaline habitats. Hemileccinum brevisporum is found in subtropical broad-leaved and mixed forests, growing with members of Fagaceae (Castanopsis calathiformis, Ca. indica, Ca. orthacantha; Lithocarpus hancei, L. mairei; Quercus griffithii) and Pinaceae (Pinus yunnanensis or P. armandii) on acidic or slightly alkaline soils. The species we described here are hardly seen in the wild mushroom market, thus their edibility is unknown yet. However, referring to the edibility of the European/American species of Hemileccinum [41][42][43][44][45]49], the newly described species could also be edible, but we need more investigations to confirm this.
Overall, the proposed new species are significantly different from the Asian species H. indecorum because the viscid pileus and stipe of the latter species are densely covered with whitish to dirty white, small conical to subconical to irregular squamules [15]. They also quite differ from the European species H. impolitum, which has a relatively bald pileal surface and a collapsed trichoderm pileipellis when mature [1]. Šutara reported that the basidiospores of H. impolitum are smooth [1]. Our re-examination of European material of H. impolitum under SEM indicated that there are irregular warts on the surface of basidiospores as with those of other species in Hemileccinum (Figure 4m-o). Accordingly, H. depilatum (Redeuilh) Šutara should also have a warty basidiospore surface.
The description of the new species also sheds new light on the recognition of the genus. The pileipellis of the species in this genus should mostly be regarded as (sub)epithelium to hyphoepithelium, because the pileipellis of most studied species here are composed of short inflated cells in the inner layer (subpellis) and filamentous hyphae in outer layer (suprapellis), with H. indecorum standing at one extremity with whitish to dirty white, small conical to subconical to irregularly shaped squamules on the pileus surface [15] and H. impolitum located at the other extremity with collapsed trichoderm pileipellis when mature [1]. The lateral stipe stratum of H. impolitum in this genus was diagnosed as the leccinoid type, predominantly anticlinally arranged hyphae ending in elements of the caulohymenium [1,3,4]. However, on the basis of the observation on our new species, this feature is not present in all species of Hemileccinum. The structure of the lateral stipe stratum is traceable in our species.
Based on the current study, we increased the species diversity of the genus Hemileccinum from Asia and reconstructed a comprehensive phylogenetic tree which included almost all known species of this genus. However, probably due to the limitations of species sampling or insufficient genetic variation of the DNA loci we used, the deep phylogenetic relationships within the genus remain unresolved. In future work, more species with de-tailed morphological observations and phylogenomic analysis will provide new evidence for these relationships.